SMRT-mediated co-shuttling enables export of class IIa HDACs independent of their CaM kinase phosphorylation sites

The Class IIa histone deacetylases (HDAC)4 and HDAC5 play a role in neuronal survival and behavioral adaptation in the CNS. Phosphorylation at 2/3 N-terminal sites promote their nuclear export. We investigated whether non-canonical signaling routes to Class IIa HDAC export exist because of their association with the co-repressor Silencing Mediator Of Retinoic And Thyroid Hormone Receptors (SMRT). We found that, while HDAC5 and HDAC4 mutants lacking their N-terminal phosphorylation sites (HDAC4(MUT), HDAC5(MUT)) are constitutively nuclear, co-expression with SMRT renders them exportable by signals that trigger SMRT export, such as synaptic activity, HDAC inhibition, and Brain Derived Neurotrophic Factor (BDNF) signaling. We found that SMRT's repression domain 3 (RD3) is critical for co-shuttling of HDAC5(MUT), consistent with the role for this domain in Class IIa HDAC association. In the context of BDNF signaling, we found that HDAC5(WT), which was more cytoplasmic than HDAC5(MUT), accumulated in the nucleus after BDNF treatment. However, co-expression of SMRT blocked BDNF-induced HDAC5(WT) import in a RD3-dependent manner. In effect, SMRT-mediated HDAC5(WT) export was opposing the BDNF-induced HDAC5 nuclear accumulation observed in SMRT's absence. Thus, SMRT's presence may render Class IIa HDACs exportable by a wider range of signals than those which simpl

Microarray profiling emphasizes transcriptomic differences between hippocampal in vivo tissue and in vitro cultures" for publication in Brain Communications.

From Crossref journal articles via Jisc Publications RouterHistory: epub 2021-07-07, issued 2021-07-07Article version: AMPublication status: PublishedAbstract Primary hippocampal cell cultures are routinely used as an experimentally accessible model platform for the hippocampus and brain tissue in general. Containing multiple cell types including neurons, astrocytes and microglia in a state that can be readily analysed optically, biochemically and electrophysiologically, such cultures have been used in many in vitro studies. To what extent the in vivo environment is recapitulated in primary cultures in an on-going question. Here we compare the transcriptomic profiles of primary hippocampal cell cultures and intact hippocampal tissue. In addition, by comparing profiles from wild type and the PrP 101LL transgenic model of prion disease, we also demonstrate that gene conservation is predominantly conserved across genetically altered lines

Extracellular Localisation of the C-Terminus of DDX4 Confirmed by Immunocytochemistry and Fluorescence-Activated Cell Sorting

Putative oogonial stem cells (OSCs) have been isolated by fluorescence-activated cell sorting (FACS) from adult human ovarian tissue using an antibody against DEAD-box helicase 4 (DDX4). DDX4 has been reported to be germ cell specific within the gonads and localised intracellularly. White et al. (2012) hypothesised that the C-terminus of DDX4 is localised on the surface of putative OSCs but is internalised during the process of oogenesis. This hypothesis is controversial since it is assumed that RNA helicases function intracellularly with no extracellular expression. To determine whether the C-terminus of DDX4 could be expressed on the cell surface, we generated a novel expression construct to express full-length DDX4 as a DsRed2 fusion protein with unique C- and N-terminal epitope tags. DDX4 and the C-terminal myc tag were detected at the cell surface by immunocytochemistry and FACS of non-permeabilised human embryonic kidney HEK 293T cells transfected with the DDX4 construct. DDX4 mRNA expression was detected in the DDX4-positive sorted cells by RT-PCR. This study clearly demonstrates that the C-terminus of DDX4 can be expressed on the cell surface despite its lack of a conventional membrane-targeting or secretory sequence. These results validate the use of antibody-based FACS to isolate DDX4-positive putative OSCs

Temporal profiling of the cortical synaptic mitochondrial proteome identifies ageing associated regulators of stability

Synapses are particularly susceptible to the effects of advancing age, and mitochondria have long been implicated as organelles contributing to this compartmental vulnerability. Despite this, the mitochondrial molecular cascades promoting age-dependent synaptic demise remain to be elucidated. Here, we sought to examine how the synaptic mitochondrial proteome (including strongly mitochondrial associated proteins) was dynamically and temporally regulated throughout ageing to determine whether alterations in the expression of individual candidates can influence synaptic stability/morphology. Proteomic profiling of wild-type mouse cortical synaptic and non-synaptic mitochondria across the lifespan revealed significant age-dependent heterogeneity between mitochondrial subpopulations, with aged organelles exhibiting unique protein expression profiles. Recapitulation of aged synaptic mitochondrial protein expression at the Drosophila neuromuscular junction has the propensity to perturb the synaptic architecture, demonstrating that temporal regulation of the mitochondrial proteome may directly modulate the stability of the synapse in vivo

Serum proteomic analysis focused on fibrosis in patients with hepatitis C virus infection

<p>Abstract</p> <p>Background</p> <p>Despite its widespread use to assess fibrosis, liver biopsy has several important drawbacks, including that is it semi-quantitative, invasive, and limited by sampling and observer variability. Non-invasive serum biomarkers may more accurately reflect the fibrogenetic process. To identify potential biomarkers of fibrosis, we compared serum protein expression profiles in patients with chronic hepatitis C (CHC) virus infection and fibrosis.</p> <p>Methods</p> <p>Twenty-one patients with no or mild fibrosis (METAVIR stage F0, F1) and 23 with advanced fibrosis (F3, F4) were retrospectively identified from a pedigreed database of 1600 CHC patients. All samples were carefully phenotyped and matched for age, gender, race, body mass index, genotype, duration of infection, alcohol use, and viral load. Expression profiling was performed in a blinded fashion using a 2D polyacrylamide gel electrophoresis/LC-MS/MS platform. Partial least squares discriminant analysis and likelihood ratio statistics were used to rank individual differences in protein expression between the 2 groups.</p> <p>Results</p> <p>Seven individual protein spots were identified as either significantly increased (α₂-macroglobulin, haptoglobin, albumin) or decreased (complement C-4, serum retinol binding protein, apolipoprotein A-1, and two isoforms of apolipoprotein A-IV) with advanced fibrosis. Three individual proteins, haptoglobin, apolipoprotein A-1, and α₂-macroglobulin, are included in existing non-invasive serum marker panels.</p> <p>Conclusion</p> <p>Biomarkers identified through expression profiling may facilitate the development of more accurate marker algorithms to better quantitate hepatic fibrosis and monitor disease progression.</p